Metabolic control of progenitor cell propagation during Drosophila tracheal remodeling

Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. Here we show that in response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling.

by Akt was eliminated in the mutant YkiS168A (Fig. 5e). Second, expression of UAS-Akt increased the number of YAP-SPARK droplets, suggesting that phosphorylation of Yki is elevated by the upregulation of Akt ( Fig. 5f,g). Finally, in accordance with the inhibitory role of insulin in Yki signaling, overexpression of Akt in L3 animals increased phosphorylation of Yki, reduced EdU incorporation , and reduced progenitor migration . In sum, these observations indicate that Akt phosphorylates Yki at Ser168 and impedes Yki-dependent processes.
> The in vivo assays are indirect, consistent with Akt acting upstream of Yki, but not definitively showing that Akt phosphorylates Yki in vivo.
Lines 297-8: were identified by RNA-seq of trachea progenitors are also high-confident gene targets identified by the Yki ChIP-seq experiment (Fig. 6e).
Reviewer #2 (Remarks to the Author): This work by Li, Y. et al. reports that, during Drosophila metamorphosis, tracheal progenitor cells are activated through Yorkie-dependent proliferation and migration, by a metabolic deficit-induced signalling process. This process, the authors claim, is regulated by insulin and the Hippo pathway.
The authors take advantage of single-cell analyses to analyse the regulation of progenitor-cell activation during a developmental period that involves major metabolic changes. During this period, the beginning of Drosophila metamorphosis, the organism is subjected to a metabolic deficiency akin to starvation. The authors identify a regulatory mechanism through which metabolic depression drives progenitor cell activation and division, underlying organ remodelling. Numerous studies point out that caloric restriction/metabolic depression, via the insulin pathway, has beneficiary effects on stem cell maintenance and tissue regeneration and making stem cells more likely to resist damage. Other studies have started discovering a growing web of interconnections between the Hippo pathway and metabolism. However, how the two pathways conjugate to allow for stem cell activation, and what consequences this has in the overall cell biology of stem cells, remains to be unraveled. In this context, the finding that metabolic depression triggers stem cell activation in connection with the Hippo pathway is a noteworthy result, which will be of significance to the fields of organogenesis, growth, ageing and regeneration. This work generated a great deal of transcriptomic data, and the questions are generally well addressed experimentally. The authors also have generated new tools which will be of significance to the field. However, I have major suggestions that will hopefully make some points clearer.

MAJOR COMMENTS
1) The figures are not very well exposed and sometimes are difficult to interpret. As a general suggestion for all figures, when one panel shows only one channel, it should be represented in gray scale and not in red or green. This makes analysis clearer. Colour panels should only be used when using colocalizations of more than one channel. It is rather awkward that scale bars are only represented in one of the panels in groups of images. I understand that is because the scale is the same for all. However, in the following cases it is not clear: 3) In many places, the manuscript fails to clearly expose the hypothesis and conclusions of the experiments. Some sentences need to be revised and rewritten. For instance: -in lines 143-144 the authors say: "This suggests that insulin pathway activity decreases in these conditions of reduced metabolism" Reduced metabolism usually goes together with insulin pathway activity decreases. In addition, PI3K activity is stimulated by diverse growth factor receptors. -in lines 175/176 the authors say: "These results suggest that insulin pathway is suppressed upon metabolic depletion." Is this really a conclusion drawn from these particular experiments? Metabolic depletion is generally a consequence of caloric restriction/insulin pathway suppression.
4) The methods section is not clear regarding some of the experiments. This should be revised. As an example, in Figure 3 the authors express Yki RNAi using a btl-GAL4 driver, but it is not clear if this was done under Gal80 conditions (the methods section does not refer to this particular RNAi under the Gal80ts section). If Gal80 was not used, does this mean YkiRNAi was expressed from embryonic stages? Also XXX 5) The authors refer to the population of tracheoblasts examined throughout this study as stem cells. However, a more correct way should be to name them progenitor cells. This should be changed throughout the whole manuscript, title included. 6) In figure 2l'' increased exlacZ expression is not clear in comparison with the L3 control. This should be quantified. These small differences could be attributed to different detection thresholds and needs an internal control. The same applies to figure 4 f-g.

7)
Chen and Krasnow reported in 2014 that tracheal progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. In this process the chemoattractant is Bnl, which is expressed dynamically ahead of the progenitors. In this work they show that Bnl knockdown abrogates progenitor outgrowth and migration. In this work the authors show that knockdown of Yki reduced progenitor migration. It is therefore essential to show if the Hippo pathway is interfering with the FGF pathway and consequently affecting migration. 8) Finally, for the sake of clarity, I suggest that the authors include a graphical model and refer to it in the discussion.
-Text should be revised for clarity.
The authors present an analysis of yki function in larval airway progenitors of the Drosophila respiratory system. They find that Insulin receptor (InR) signaling intersects with yki activation to control the proliferation of and migration of larval tracheoblasts. They further use ChIP experiments and RNA sequencing to define targets of yki at the relevant tiem points of pupal development and go on to validate some of the targets in the process of tracheoblast division and migration. Overall, the manuscript provides considerable novelty describing the regulation of tracheoblasts and and presenting new reagents for in situ detection of InR, ATK and AMPK activation. I have some concerns on the coincidence of Inr a ctvationof the interpretation of the "cell migration" phenotypes, the analysis and presentation of the ChIP-seq experiment and the reliability if the SPARK-Yap reporter.
1) Presentation of reporters in the dorsal trunk cell instead of tracheoblasts. Lack of conisidense of GFP signals with ex-lacZ expression. Fig.2 shows convinsingly that InR is active in the larval dorsal trunk cells and this activity is reduced in early pupae. The DT cells are destined to die (Chen and Krasnow 2014). The activity of the reporters si not shown in the tracheoblasts. How do the reporter dots differ in tracheoblasts, where exlacZ is increased? Fig 2L, 2LL'' It should be possible to do double stainings for GFP and LacZ in dissected larval and early pupal trachea. On this context it would be helpful for the reader to explain how the differences in reporter dot number and size between panels 2I and 2I''are interpreted.
2) Cell migration phenotypes. The authors visualize groups of tracheoblasts with a cytoplasmic marker moe-RFP and measure how far the stainings extend on a metamere. Any intervention with the cytoskeleton (not necessarily migration) would extend or diminish (contract) the RFP staining. To state an effect on migration the authors would need to label and track single tracheoblast or do longer imaging (like in figure 1) to allow tracheobalst to extend into more posterior metameres. In figure 4 h,I, I have difficulties to see and interpret the differences in YAP-SPARK dots. exlacZ is clearly upon insulin addition is it possible to do a double staining for LacZ and GFP here? Figure 4j-q needs quantifications. Addition of 20E stimulates cell extensions but the effect of insulin on it is not visible to me. How was the quantification in 4m done? How many animals and how many metameres were counted? What does % of metamere mean? Fig5 K-l'' and 5 mSimilarly for 7J.
3) ChIP seq assay and presentation. How were the ChIP signals quantified relatively to the IgG control? The minimum would be to show the igG tracks below the tracks in Fig6 using the same scales and describe how the data were normalized. 4) The specificity of the Yki reporter is questionable in vivo. Extended data figure 3 The dots are smaller in P compared to O but they are more in P compared to O. In other figures like Fig 4I more dots are taken to indicate stronger signaling. We need to see some type of reliable quantification.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this study, Li and colleagues show that during metamorphosis, InR signaling activates tracheoblast proliferation and migration through modifying Yki activity. The authors show that metabolic activity in the trachea is depressed during puparation and identify metabolic genes and Hippo and Hippo pathway genes as those differentially expressed between larval and pupal datasets. In comparing fasting and normal fed L3 larval tracheoblast gene expression, InR/PI3K and Hippo pathways were among those showing the most significant changes. Using a InR pathway activity reporter, InR activity was seen to decline during puparium formation. Reporters of InR, Akt and AMP kinase activity were generated and used to follow activity in tracheoblasts. InR pathway activity was found to be suppressed upon metabolic depletion. A Hippo pathway reporter (expanded lacZ expression) was likewise examined and found to increase during the larval-pupal transition and under starvation conditions. Proliferation and migration were decreased by Yki RNAi, while Warts RNAi or Yki overextression resulted in increased proliferation. Expression of a dominant negative insulin receptor inhibited Yki activity as assessed by the ex-lacZ reporter. A YAP/Yki reporter was generated and was also used to support this finding. Akt activity is regulated by InR and the authors found that Akt is able to phosphorylate and inactivate Yki in vitro. Authors show additional data consistent with an in vivo role of Akt in Yki phosphorylation. Authors identify candidate Yki target genes in tracheoblasts based on ChIP analysis, and show that yki RNAi downregulates expression of these candidates. RNAi analysis of yki regulated genes identify some that affect proliferation and others that affect migration.
All together, these results demonstrate an important interaction between InR and Hippo pathways that regulate tracheoblast behavior during metamorphosis. The data is largely compelling and the manuscript should be accepted with minor revisions. Authors should address the following: Authors' response: We thank this reviewer for acknowledging the significance and quality of our work.
Lines 93-5: The larval tracheal network consists of bilateral dorsal trunk (DT) tubes which are linked by dorsal branches (DBs), transverse connectives (TCs), and spiracular branches (SBs) in each of the 10 tracheal metameres (Tr1-Tr10; Fig. 1a,b, arrows) 31 > SB and TC do not link DTs...and a number of tracheal branches in the larval network are omitted (eg lateral trunk, visceral branch, ganglionic branch).
Authors' response: We thank this reviewer for pointing out this omission in our previous submission. We have revised Fig. 1a to include all the relevant branches as suggested by this reviewer.
Lines 96-9: Clusters of tracheal progenitors are present in the 4th and 5th of the ten bilaterally symmetric SBs, and are visible in Fig. 1b by the fluorescence of red fluorescent protein (RFP) that was expressed by a transgene containing a promoter fragment that is specific for tracheal progenitors 32,33 > Ref 32 and 33 describe a btl enhancer element that is not specific for progenitors so far as reported in these refs, but is used by the authors to label the entire tracheal system; authors should clarify.
Authors' response: We thank this reviewer for pointing out this issue that was not adequately explained in our previous submission. In our study, we used a P[B123]-RFP-moe allele that harbors part of btl enhancer and marks activated progenitor cells 1 . This line was reported in the Chen and Krasnow paper. In our revised manuscript we cite this paper specifically when describing our experimental setup, and we apologize for our previous error.
Lines 150-2: Phase separation and formation of droplets are achieved by the combination of multivalency and kinase activity-dependent (PPI).
> word for which PPI is an abbreviation (protein-protein interaction) is missing.
Authors' response: We made the suggested change.
Lines 167-9: GFP droplets were abundant in L3 trachea of the three reporter lines, but not in animals with of reduced function of the respective kinases (Extended Data Fig. 3i-n).
> delete of from "with of reduced" Authors' response: We made the suggested change.
Lines 258-67: Phosphorylation of FLAG-Yki was detected by an antibody that recognizes phospho-Akt substrates, but not by an antibody against phospho-AMPK substrates (Fig. 5d). Phosphorylation of Yki by Akt was eliminated in the mutant YkiS168A (Fig. 5e). Second, expression of UAS-Akt increased the number of YAP-SPARK droplets, suggesting that phosphorylation of Yki is elevated by the up-regulation of Akt (Fig. 5f,g). Finally, in accordance with the inhibitory role of insulin in Yki signaling, overexpression of Akt in L3 animals increased phosphorylation of Yki, reduced EdU incorporation (Fig. 5h-j), and reduced progenitor migration (Fig. 5k-m). In sum, these observations indicate that Akt phosphorylates Yki at Ser168 and impedes Yki-dependent processes.
> The in vivo assays are indirect, consistent with Akt acting upstream of Yki, but not definitively showing that Akt phosphorylates Yki in vivo.
Authors' response: We agree with this reviewer and have revised the text accordingly (see page 11).
Lines 297-8: were identified by RNA-seq of trachea progenitors are also high-confident gene targets identified by the Yki ChIP-seq experiment (Fig. 6e).

> high confidence?
Authors' response: We made the suggested correction.

Reviewer #2 (Remarks to the Author):
This work by Li, Y. et al. reports that, during Drosophila metamorphosis, tracheal progenitor cells are activated through Yorkie-dependent proliferation and migration, by a metabolic deficit-induced signalling process. This process, the authors claim, is regulated by insulin and the Hippo pathway.
The authors take advantage of single-cell analyses to analyse the regulation of progenitor-cell activation during a developmental period that involves major metabolic changes. During this period, the beginning of Drosophila metamorphosis, the organism is subjected to a metabolic deficiency akin to starvation. The authors identify a regulatory mechanism through which metabolic depression drives progenitor cell activation and division, underlying organ remodelling. Numerous studies point out that caloric restriction/metabolic depression, via the insulin pathway, has beneficiary effects on stem cell maintenance and tissue regeneration and making stem cells more likely to resist damage. Other studies have started discovering a growing web of interconnections between the Hippo pathway and metabolism. However, how the two pathways conjugate to allow for stem cell activation, and what consequences this has in the overall cell biology of stem cells, remains to be unraveled. In this context, the finding that metabolic depression triggers stem cell activation in connection with the Hippo pathway is a noteworthy result, which will be of significance to the fields of organogenesis, growth, ageing and regeneration.
Authors' response: We thank this reviewer for his/her recognition of the significance of our work.
This work generated a great deal of transcriptomic data, and the questions are generally well addressed experimentally. The authors also have generated new tools which will be of significance to the field. However, I have major suggestions that will hopefully make some points clearer.

MAJOR COMMENTS
1) The figures are not very well exposed and sometimes are difficult to interpret. As a general suggestion for all figures, when one panel shows only one channel, it should be represented in gray scale and not in red or green. This makes analysis clearer. Colour panels should only be used when using colocalizations of more than one channel. It is rather awkward that scale bars are only represented in one of the panels in groups of images. I understand that is because the scale is the same for all. However, in the following cases it is not clear :  Fig 2 h-j  Fig 3 a-c Authors' response: As suggested by this reviewer, we changed one-channel images to grey images, especially images showing SPARK reporters. We left images containing btl>GFP or P[B123]-RFPmoe in color for comparison in analysis. We added scale bars for Fig. 2 and Fig. 3 as suggested by this reviewer.
2) Panels in Fig. 7 I' and I'' seem to be the same. This should be checked and changed to the appropriate panel.
Authors' response: We thank this reviewer for pointing this out and we have made appropriate replacements.
3) In many places, the manuscript fails to clearly expose the hypothesis and conclusions of the experiments. Some sentences need to be revised and rewritten. For instance: -in lines 143-144 the authors say: "This suggests that insulin pathway activity decreases in these conditions of reduced metabolism" Reduced metabolism usually goes together with insulin pathway activity decreases. In addition, PI3K activity is stimulated by diverse growth factor receptors. -in lines 175/176 the authors say: "These results suggest that insulin pathway is suppressed upon metabolic depletion." Is this really a conclusion drawn from these particular experiments? Metabolic depletion is generally a consequence of caloric restriction/insulin pathway suppression.
Authors' response: We thank this reviewer for his/her suggestions. We have made significant changes to enhance the presentation of our work. Several sections were completely rewritten in the revised manuscript as highlighted in red.
4) The methods section is not clear regarding some of the experiments. This should be revised. As an example, in Figure 3 the authors express Yki RNAi using a btl-GAL4 driver, but it is not clear if this was done under Gal80 conditions (the methods section does not refer to this particular RNAi under the Gal80ts section). If Gal80 was not used, does this mean YkiRNAi was expressed from embryonic stages? Also XXX Authors' response: The expression of ykiRNAi is under the control of tub-Gal80 ts . We apologize for not adequately describing the experimental setup in our previous submission. We have rewritten the Methods section as suggested by the reviewer.
5) The authors refer to the population of tracheoblasts examined throughout this study as stem cells. However, a more correct way should be to name them progenitor cells. This should be changed throughout the whole manuscript, title included.
Authors' response: We revised it throughout the entire manuscript accordingly. 6) In figure 2l'' increased exlacZ expression is not clear in comparison with the L3 control. This should be quantified. These small differences could be attributed to different detection thresholds and needs an internal control. The same applies to figure 4 f-g.
Authors' response: With regard to this reviewer's suggestion of internal control, we utilized btl-Gal4-driven UAS-GFP that serves as an internal control. The GFP signal is independent of these treatments. We performed quantitative analysis of ex-lacZ expression, as shown in Fig. 2p and Fig.  4j. The procedure is described in the Methods section. 7) Chen and Krasnow reported in 2014 that tracheal progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. In this process the chemoattractant is Bnl, which is expressed dynamically ahead of the progenitors. In this work they show that Bnl knockdown abrogates progenitor outgrowth and migration. In this work the authors show that knockdown of Yki reduced progenitor migration. It is therefore essential to show if the Hippo pathway is interfering with the FGF pathway and consequently affecting migration.
Authors' response: According to this reviewer's suggestion, we perturbed FGF pathway by expressing a dominant negative form of FGFR, Btl DN in the trachea. Consistent with Chen and Krasnow, our results showed that perturbation of Btl abolished the migration of progenitors ( Supplementary Fig. 11d,e and Supplementary Movie 4), which phenocopied knockdown of Yki. In addition, expression of btl DN decreased activity of Yki in the trachea (See Supplementary Fig. 11ac). These results provide further support an interplay between FGF pathway and Yki signaling in tracheal progenitor migration. 8) Finally, for the sake of clarity, I suggest that the authors include a graphical model and refer to it in the discussion.
Authors' response: According to this reviewer's suggestion, we added a signaling network diagram in the discussion (Supplementary Fig. 10).
-In the methods section, statistic methods are only briefly described and only for the EdU experiments.
-Text should be revised for clarity. Authors' response: According to this reviewer's suggestion, we added statistic and quantification methods for staining, SPARK signal, and progenitor migration in Methods section of our revised manuscript.
Reviewer #3 (Remarks to the Author): The authors present an analysis of yki function in larval airway progenitors of the Drosophila respiratory system. They find that Insulin receptor (InR) signaling intersects with yki activation to control the proliferation of and migration of larval tracheoblasts. They further use ChIP experiments and RNA sequencing to define targets of yki at the relevant time points of pupal development and go on to validate some of the targets in the process of tracheoblast division and migration. Overall, the manuscript provides considerable novelty describing the regulation of tracheoblasts and and presenting new reagents for in situ detection of InR, AKT and AMPK activation. I have some concerns on the coincidence of InR activation of the interpretation of the "cell migration" phenotypes, the analysis and presentation of the ChIP-seq experiment and the reliability if the SPARK-Yap reporter. Authors' response: We thank this reviewer for recognizing the novelty of our work.
1) Presentation of reporters in the dorsal trunk cell instead of tracheoblasts. Lack of coincidence of GFP signals with ex-lacZ expression. Fig.2 shows convincingly that InR is active in the larval dorsal trunk cells and this activity is reduced in early pupae. The DT cells are destined to die (Chen and Krasnow 2014). The activity of the reporters is not shown in the tracheoblasts. How do the reporter dots differ in tracheoblasts, where exlacZ is increased? Fig 2L, 2LL'' It should be possible to do double stainings for GFP and LacZ in dissected larval and early pupal trachea. On this context it would be helpful for the reader to explain how the differences in reporter dot number and size between panels 2I and 2I''are interpreted.
Authors' response: Regarding to the reviewer's suggestion of InR signal in tracheoblasts, we performed double staining of tracheoblasts for GFP and lacZ. The results showed that the signal of InR-SPARK reporter inversely correlated with the expression of ex-lacZ (Fig. 2n,o). Consistent with InR-SPARK in DT (Fig. 2i,i'), the number and size of GFP droplets in tracheoblast was reduced in white pupae, compared with L3 larvae. We calculated the ratio of pixel fluorescence intensity from cellular droplets relative to total pixel intensity of the cell, which represents the strength of SPARK signal (Fig. 2q). Detailed description is added in the text. The formula is provided in the Methods section.
2) Cell migration phenotypes. The authors visualize groups of tracheoblasts with a cytoplasmic marker moe-RFP and measure how far the stainings extend on a metamere. Any intervention with the cytoskeleton (not necessarily migration) would extend or diminish (contract) the RFP staining. To state an effect on migration the authors would need to label and track single tracheoblast or do longer imaging (like in figure 1) to allow tracheoblast to extend into more posterior metameres. In figure 4 h,I, I have difficulties to see and interpret the differences in YAP-SPARK dots. exlacZ is clearly upon insulin addition is it possible to do a double staining for LacZ and GFP here? Figure  4j-q needs quantifications. Addition of 20E stimulates cell extensions but the effect of insulin on it is not visible to me. How was the quantification in 4m done? How many animals and how many metameres were counted? What does % of metamere mean? Fig5 K-l'' and 5 m Similarly for 7J.
Authors' response: We agree with this reviewer that time-lapse imaging is favorable for analysis of migration. We provide steady images together with 5-hr movies (see Supplementary Movie 1 for Figure 1 and Supplementary Movie 3 for other Figures), as tracking single tracheoblast through intact cuticle is technically challenging. We performed double staining of ex-lacZ and YAP-SPARK, according to this reviewer's suggestion. It should be mentioned that the GFP droplets are liquid-like condensates and fixation impairs the fluorescent signal as well as the background fluorescence. The results showed that signal of YAP-SPARK was increased accompanying with the reduced expression of ex-lacZ upon the addition of insulin (Fig. 4h-k). We provided quantification for Figure 5a-f corresponding to previous Fig. 4j-o, as shown in Fig. 5g. For previous Fig. 4p,q (current Fig. 5h,i), we identified 18 larvae showing precocious migration of tracheal progenitors in 20 animals expressing InR DN . For analysis of progenitor migration, we examined at a minimum 8 metameres in 8 animals for each condition. % of metamere represents migration distance of progenitors relative to the length of a metamere (see Supplementary Fig. 12). The velocity of migration is calculated as the % of tracheal metamere per min. The detailed methodology for measurement is shown in Supplementary Fig. 12 and Methods section. Statistics are shown in Source Data file. We conducted this measurement for previous Fig. 5k-l'',m (current Fig. 6l-n) and Fig. 7j (current Fig. 8j) and quantification is shown in Fig. 6n and Fig. 8j.
3) ChIP seq assay and presentation. How were the ChIP signals quantified relatively to the IgG control? The minimum would be to show the igG tracks below the tracks in Fig6 using the same scales and describe how the data were normalized.